This invention relates to a method of producing a taxane-type diterpene including taxol which is useful as a therapeutic agent for ovarian cancer, mammary cancer, lung cancer and the like, and a method of obtaining cultured cells which produce the taxane-type diterpene at a high rate.
Taxol, which is useful as a therapeutic agent for ovarian cancer, mammary cancer, lung cancer and the like, is a taxane-type diterpene identified after being isolated from Taxus brevifolia NUTT, which is a plant belonging to genus Taxus, family Taxaceae and has a complex ester group which is related to its activity. Taxol can be found in all the parts of the plant body of Taxus brevifolia NUTT, but the bark has been reported to exceed all others in its content of the taxol. At present, taxol is collected from a natural or a cultivated plant body, however, the plant belonging to genus Taxus grows slowly, and it takes more than 10 years to grow to a height of 20 cm above the ground. Also the tree dies after its bark is taken off, thus it has been difficult to easily obtain a large amount of taxol. It would be advantageous if a taxane-type diterpene such as taxol and baccatin III which is a precursor of taxol, can be produced by the use of tissue culture, since a large amount of taxol can be easily obtained without cutting down the trees.
As a conventional method of producing taxol by utilizing cultured plant cells, a US patent was issued on a production method utilizing cultured cells of Taxus brevifolia NUTT (U.S. Pat. No. 5,019,504), however, the yield of taxol production described therein is 1-3 mg/l, and that is insufficient for the industrial production. Also, the production of taxol by the cell culture is unstable and even when a primary cell of high productivity can be obtained by selection, it is difficult to keep its content by subculturing [E. R. M. Wickremesine et al., World Congress on Cell and Tissue Culture (1992)].
On the other hand, as a prior art in the taxol production, a semisynthetic method from baccatin III, which is a precursor in biosynthesis of taxol, is disclosed in the specification of U.S. Pat. No. 5,015,744 issued to Holton et al. By the use of the plant tissue culture, a raw material for the semisynthetic process such -as baccatin III can be produced, thus the plant tissue culture can be also utilized for taxol production by the above-mentioned semisynthetic process.
The first object of the present invention is to provide a simple method of producing a taxane-type diterpene by plant tissue culture.
The second object of the present invention is to provide a method of obtaining cultured cells which produce a taxane-type diterpene at a high rate.
The first invention of the present application is a method of producing a taxane-type diterpene wherein a tissue or a cell of a plant which produces a taxane-type diterpene is cultured in the presence of at least one substance selected from the group consisting of jasmonic acids, compounds containing a heavy metal, complex ions containing a heavy metal, heavy metal ions, amines and antiethylene agents, then the taxane-type diterpene is recovered from the resulting cultures.
The second invention of the present application is a method of producing a taxane-type diterpene wherein a tissue or a cell of a plant which produces a taxane-type diterpene is cultured by controlling the oxygen concentration in a gas phase in a culture vessel to less than the oxygen concentration in the atmosphere, from the initial stage of the culture, or by controlling the dissolved oxygen concentration in a fluid medium which is in contact with the tissue or the cell to less than the saturated dissolved oxygen concentration at that temperature, from the initial stage of the culture, then the taxane-type diterpene is recovered from the resulting cultures.
The third invention of the present application is a method of obtaining cultured cells which produce a taxane-type diterpene at a high rate, wherein cells of a plant which produces a taxane-type diterpene are fractionated into a plurality of layers according to the difference in their specific gravities, and cells contained in at least one layer are cultured, then such cultured cells that produce the taxane-type diterpene at a high rate are selected from among those cultured cells.
The present invention will be described in further detail.
The taxane-type diterpene, which is an object for the present invention, is not particularly limited to any diterpene as far as it has a taxane skeleton, and the illustrative examples include taxol, 7-epitaxol, baccatin III, 7-epibaccatin III, cephalomannine, 7-epicephalomannine, 10-deacetylbaccatin III, 10-deacetylcephalomannine, 10-deacetyltaxol, taxagifine, an analogue thereof, taxane 1a, an analogue thereof, xylosyl cephalomannine, xylosyl taxol and the like.
Examples of the plant to be used in the present invention which produces the taxane-type diterpene are those belonging to genus Taxus, such as Taxus baccata LINN, Taxus cuspidata SIEB. et ZUCC, Taxus cuspidata SIEB. et ZUCC var. nana REHDER, Taxus brevifolia NUTT, Taxus canadiensis MARSH, Taxus chinensis, and Taxus media.
According to the first invention of the present application, culture of the above-mentioned plant can be carried out by the previously known method except that the tissue or the cell of the plant which produces the taxane-type diterpene is cultured in the presence of at least one substance selected from the group consisting of jasmonic acids, compounds containing a heavy metal, complex ions containing a heavy metal, heavy metal ions, amines and antiethylene agents.
Examples of jasmonic acids, which are objects for the first invention of the present application, include a compound represented by the general formula (I): 
[wherein, R1a, R1b, R1c, R1d, R1e and R1f respectively represent hydrogen atom, hydroxyl group, alkyl group having 1 to 6 carbon atoms, or alkoxy group having 1 to 6 carbon atoms;
R2, R3, R4, R5 and R6a respectively represent hydrogen atom or alkyl group having 1 to 6 carbon atoms;
a side chain consisting of C1xe2x80x94C2xe2x80x94C3xe2x80x94C4xe2x80x94C5xe2x80x94C6 may contain one or more double bonds;
R6b represents hydroxyl group or xe2x80x94Oxe2x80x94carbohydrate residue;
R7 represents hydroxyl group, OM (wherein M is alkali metal atom, alkaline earth metal atom or NH4), NHR8 (wherein R8 represents hydrogen atom, acyl group having 1 to 6 carbon atoms, alkyl group having 1 to 6 carbon atoms or amino acid residue), OR9 (wherein R9 is alkyl group having 1 to 6 carbon atoms or carbohydrate residue), or alkyl group having 1 to 6 carbon atoms;
n is an integer of 1-7;
and in the above-mentioned five-membered ring, a double bond may be formed between the neighboring member carbon atoms],
a compound represented by the general formula (II): 
[wherein, R1a, R1b, R1c, R1d, R1e and R1f respectively represent hydrogen atom, hydroxyl group, alkyl group having 1 to 6 carbon atoms, or alkoxy group having 1 to 6 carbon atoms;
R2, R3, R4, R5 and R6 respectively represent hydrogen atom or alkyl group having 1 to 6 carbon atoms;
a side chain consisting of C1xe2x80x94C2xe2x80x94C3xe2x80x94C4xe2x80x94C5xe2x80x94C6 may contain one or more double bonds;
R7 represents hydroxyl group, OM (wherein M is alkali metal atom, alkaline earth metal atom or NH4), NHR8 (wherein R8 represents hydrogen atom, acyl group having 1 to 6 carbon atoms, alkyl group having 1 to 6 carbon atoms or amino acid residue), OR9 (wherein R9 is alkyl group having 1 to 6 carbon atoms or carbohydrate residue), or alkyl group having 1 to 6 carbon atoms;
n is an integer of 1-7;
and in the above-mentioned five-membered ring, a double bond may be formed between the neighboring member carbon atoms],
and a compound represented by the general formula (III): 
[wherein, R1a, R1b, R1c, R1d, R1e and R1f respectively represent hydrogen atom, hydroxyl group, alkyl group having 1 to 6 carbon atoms, or alkoxy group having 1 to 6 carbon atoms;
R2, R3, R4, R5 and R6 respectively represent hydrogen atom or alkyl group having 1 to 6 carbon atoms;
a side chain consisting of C1xe2x80x94C2xe2x80x94C3xe2x80x94C4xe2x80x94C5xe2x80x94C6 may contain one or more double bonds;
R7 represents hydroxyl group, OM (wherein M is alkali metal atom, alkaline earth metal atom or NH4), NHR8 (wherein R8 represents hydrogen atom, acyl group having 1 to 6 carbon atoms, alkyl group having 1 to 6 carbon atoms or amino acid residue), OR9 (wherein R9 is alkyl group having 1 to 6 carbon atoms or carbohydrate residue), or alkyl group having 1 to 6 carbon atoms;
n is an integer of 1-7;
and in the above-mentioned five-membered ring, a double bond may be formed between the neighboring member carbon atoms].
Preferable examples of jasmonic acids represented by the above-mentioned general formula (I) include a compound represented by the general formula (Ixe2x80x2): 
[wherein, R1 represents hydrogen atom or hydroxyl group;
a side chain consisting of C1xe2x80x94C2xe2x80x94C3xe2x80x94C4xe2x80x94C5xe2x80x94C6 may contain a double bond between C1 and C2, between C2 and C3, or between C3 and C4;
R6b represents hydroxyl group or xe2x80x94Oxe2x80x94carbohydrate residue;
R7xe2x80x2 represents hydroxyl group, OM (wherein M is alkali metal atom, alkaline earth metal atom or NH4), NHR8 (wherein R8xe2x80x2 represents hydrogen atom, acyl group having 1 to 4 carbon atoms, alkyl group having 1 to 4 carbon atoms or amino acid residue) or OR9xe2x80x2 (wherein R9xe2x80x2 represents alkyl group having 1 to 4 carbon atoms or carbohydrate residue);
n is an integer of 1-7;
and in the above-mentioned five-membered ring, a double bond may be formed between the neighboring member carbon atoms], and preferable examples of jasmonic acids represented by the above-mentioned general formula (II) include a compound represented by the general formula (IIxe2x80x2): 
[wherein, R1xe2x80x2 represents hydrogen atom or hydroxyl group;
a side chain consisting of C1xe2x80x94C2xe2x80x94C3xe2x80x94C4xe2x80x94C5xe2x80x94C6 may contain a double bond between C1 and C2, between C2 and C3, or between C3 and C4;
R7xe2x80x2 represents hydroxyl group, OM (wherein M is alkali metal atom, alkaline earth metal atom or NH4), NHR8xe2x80x2 (wherein R8xe2x80x2 represents hydrogen atom, acyl group having 1 to 4 carbon atoms, alkyl group having 1 to 4 carbon atoms or amino acid residue) or OR9xe2x80x2 (wherein R9xe2x80x2 represents alkyl group having 1 to 4 carbon atoms or carbohydrate residue);
n is an integer of 1-7;
and in the above-mentioned five-membered ring, a double bond may be formed between the neighboring member carbon atoms], and preferable examples of jasmonic acids represented by the above-mentioned general formula (III) include a compound represented by the general formula (IIIxe2x80x2), 
[wherein, R1xe2x80x2 represents hydrogen atom or hydroxyl group;
a side chain consisting of C1xe2x80x94C2xe2x80x94C3xe2x80x94C4xe2x80x94C5xe2x80x94C6 may contain a double bond between C1 and C2, between C2 and C3, or between C3 and C4;
R7xe2x80x2 represents hydroxyl group, OM (wherein M is alkali metal atom, alkaline earth metal atom or NH4), NHR8xe2x80x2 (wherein R8xe2x80x2 represents hydrogen atom, acyl group having 1 to 4 carbon atoms, alkyl group having 1 to 4 carbon atoms or amino acid residue) or OR9xe2x80x2 (wherein R9xe2x80x2 represents alkyl group having 1 to 4 carbon atoms or carbdhydrate residue);
n is an integer of 1-7;
and in the above-mentioned five-membered ring, a double bond may be formed between the neighboring member carbon atoms].
In the above-mentioned general formulae (I), (II) and (III), examples of alkyl group having 1 to 6 carbon atoms represented by R1a, R1b, R1c, R1d, R1e, R1f, R2, R3, R4, R5, R6, R6a, R7, R8 or R9 include, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, and n-hexyl groups.
In the above-mentioned general formulae (I), (II) and (III), examples of alkoxy group having 1 to 6 carbon atoms represented by R1a, R1b, R1c, R1d, R1e or R1f include, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, t-butoxy, n-pentyloxy and n-hexyloxy groups.
When R7 is OM, examples of an alkali metal atom or an alkaline earth metal atom represented by M include, for example, sodium, potassium and calcium.
When R7 is NHR8, the acyl group having 1 to 6 carbon atoms represented by R8 may have either a straight chain or a branched chain, and their examples include, for example, formyl, acetyl, propionyl, butyryl, valeryl, hexanoyl and acryloyl groups.
When R7 is NHR8, examples of an amino acid residue represented by R8 include isoleucyl, tyrosyl, and tryptophyl groups.
When R7 is OR9, an example of a carbohydrate residue represented by R9 is glucopyranosyl group, and when R6b is xe2x80x94Oxe2x80x94carbohydrate residue in the above-mentioned general formula (I), an example of a carbohydrate residue is glucopyranosyl group.
In the compounds represented by the general formulae (I), (II) and (III), a double bond may be formed between the neighboring member carbon atoms in the five-membered ring.
Illustrative examples of the compound represented by the general formula (I) include those shown as follows; 
Illustrative examples of the compound represented by the general formula (II) include those shown as follows; 
Illustrative examples of the compound represented by the general formula (III) include those shown as follows;
(Compound I)
R1a, R1b, R1c, R1d, R1e, R1f, R2, R3, R4, R5, R6: H
A double bond is formed between C3 and C4.
R7: xe2x80x94OH or xe2x80x94OCH3 
n: 1 to 3
(Compound J)
R1a, R1b, R1c, R1d, R1e, R1f, R2, R3, R4, R5, R6: H
R7: xe2x80x94OH
n: 1
Illustrative examples of the compound represented by the general formula (III) wherein R1a, R1b, R1c, R1d, R1e or R1f is hydroxyl group, or a double bond is formed between the neighboring member carbon atoms in the five-membered ring, include those shown as below; 
Preferable examples of a compound represented by the general formula (I), (II) or (III) include the compounds wherein R1a, R1b, R1c, R1d, R1e, R1f, R2, R3, R4, R5 and R6 are hydrogen atoms, R7 is hydroxyl group or methoxy group, and a side chain consisting of C1xe2x80x94C2xe2x80x94C3xe2x80x94C4xe2x80x94C5xe2x80x94C6 does not contain a double bond, or contain a double bond between C1 and C2, between C2 and C3, or between C3 and C4.
Jasmonic acids to be used in the present invention which are represented by the general formula (I), (II) or (III) have various stereoisomers (cis-trans isomers and optical isomers), and each isomer can be used alone or in the form of a mixture.
All of the jasmonic acids shown above have the effect of improving the productivity in the taxane-type diterpene production, however, tuberonic acid, methyl tuberonate, cucurbic acid, methyl cucurbate, jasmonic acid and methyl jasmonate, which are the compounds represented by the general formula (I), (II) or (III) wherein R1a, R1b, R1c, R1d, R1e, R1f, R2, R3, R4, R5 and R6 are hydrogen atoms, R7 is hydroxyl group or methoxy group, n is 1, and a double bond is formed between C3 and C4, are particularly preferable from the view point of their high effectiveness in improving the productivity.
These jasmonic acids are prepared by synthesis or extraction and the like from a plant (H. Yamane et al: Agric. Biol. Chem., 44, 2857-2864(1980)).
By the way, there is a description teaching that jasmonic acids are produced by various plants by themselves as a phytohormone-like substance which induces various reactions related to growth promotion, maturation of tissue and appearance of resistance to disease (Teruhiko Yoshihara. Shokubutsu Saib{overscore (o)} K{overscore (o)}gaku, Vol 2, No.4 523-531 (1990)).
Accordingly, the jasmonic acids involved in the present invention, can be not only added from outside of the culture system, but also produced by the cultured cells or cultured tissues by themselves. An example of a method to promote the production of such endogenous jasmonic acids by the cultured cells or cultured tissues includes addition of microorganism cultures, an extract or a heat-treated substance thereof, or a plant extract to a culture medium, and an illustrative example of such a method is a process of adding a fungus cell wall fraction described by M. J. Mueller et al., Proc. Natl. Acad. Sci. U.S.A., 90 (16), 7490-7494 (1993)). It is also possible to increase the amount of the produced endogenous jasmonic acid by partially damaging cultured cells or cultured tissues mechanically, or with ultraviolet rays, or heat, and one illustrative example of such a process is mechanical cytoclasis of a part of cells (R. A. Cleeman et al., Proc. Natl. Acad. Sci. U.S.A., 89(11), 4938-4941 (1989).
Since jasmonic acids are hardly soluble in water, they are usually dissolved in an organic solvent such as ethanol and methanol or dissolved in a surfactant and the like, then added to a culture medium. Jasmonic acids in liberated form can be used as they are, or they are used in the form of a salt by being neutralized with an alkali.
Of the jasmonic acids, those compounds represented by the formula (I) or (III) tend to be in stable trans-form rather than unstable cis-form, since epimerization occurs at the alpha-position to the carbonyl group in the five-membered ring by an acid, an alkali or heat. In an equilibrium experiment utilizing natural or synthesized jasmonic acids, the trans-form is present in the ratio of 90% and the cis-form is present in the ratio of 10%. Generally, cis-form is considered to have a higher activity, but jasmonic acids to be used in the present invention include all the stereoisomers of the compounds represented by the above-mentioned formula (I) or (III), and the mixture thereof.
Jasmonic acids are required to have a concentration in a culture medium of 0.01-1000 xcexcM, and it is particularly preferable, according to the first invention of the present application, to control the concentration of the jasmonic acids to be in the range of 0.1 to 500 xcexcM.
Induction of some secondary metabolites by addition of jasmonic acids to plant cell cultures is described in DE 4122208 C1, however, there have been no reports on carrying out tissue culture of a taxane-type diterpene producing plant in the presence of jasmonic acids as a medium additive, and it has been beyond all expectations that the amount of the produced taxane-type diterpene, which has totally different biosynthetic pathway or biosynthesis controlling mechanism from those of the secondary metabolites disclosed in the above-mentioned patent, was increased by the method of the first invention of the present application.
There is a description in the International Publication WO No. 93/17121 that jasmone or methyl jasmone, which has a structure analogous to those of jasmonic acids represented by the formula (I), (II) or (III) to be used in the present invention, is effective in induction of taxol production. However, these compounds do not have a group such as carboxyl, which is represented by the formula: xe2x80x94(CH2)nxe2x80x94COxe2x80x94R7 in the formula (I), (II) or (III), unlike the jasmonic acids, and the taxol inducing activity of these compounds was found to be low (see Comparative Example No. 24).
The heavy metals, which are objects for the first invention of the present application, are not particularly limited to any heavy metal as far as it belongs to the copper group or the iron group, however, as the metal belonging to the copper group, the use of silver is particularly preferable, and as the metal belonging to the iron group, the use of cobalt is particularly preferable. In addition to that, when silver or cobalt is used, it is preferably used in the form of a compound containing the said heavy metal, a complexion containing the said metal or in the form of the said metal ion. These compounds can be used alone or in combination.
Illustrative examples of the compound containing silver include silver nitrate, silver sulfate, silver fluoride, silver chlorate, silver perchlorate, silver acetate, silver sulfite, silver hexafluorophosphate(V), silver tetrafluoroborate, diamine silver(I) sulfate, potassium diaminoargentate(I) and the like. Among these, particularly preferable compounds can be exemplified by silver nitrate, silver sulfate and the like.
Illustrative examples of the complex ion containing silver include [Ag(S2O3)2]3xe2x88x92, [Ag(S2O3)3]5xe2x88x92, [Ag(NH3)2]+, [Ag(CN)2]xe2x88x92, [Ag(CN)3]2xe2x88x92, [Ag(SCN)2]xe2x88x92, [Ag(SCN)4]3xe2x88x92 and the like. Among these, particularly preferable complex ions can be exemplified by [Ag(S2O3)2]3xe2x88x92, [Ag(S2O3)3]5xe2x88x92 and the like.
Illustrative examples of the compound containing cobalt include cobalt chloride, cobalt nitrate, cobalt sulfate, cobalt fluoride, cobalt perchlorate, cobalt bromide, cobalt iodide, cobalt selenate, cobalt thiocyanate, cobalt acetate, ammonium cobalt sulfate, cobalt(II) potassium sulfate, hexaamminecobalt(III) chloride, pentaammineaquacobalt(III)chloride, nitropentaamminecobalt(III) chloride, dichlorotetraamminecobalt(III) chloride hemihydrate, dinitrotetraamminecobalt(III) chloride, carbonatotetraamminecobalt(III) chloride, ammonium tetranitrodiamminecobaltate(III), sodium hexanitrocobaltate(III), tris(ethylenediamine)cobalt(III) chloride trihydrate, dichlorobis(ethylenediamine)cobalt(III) chloride, potassium tris(oxalato)cobaltate(III) trihydrate, potassium hexacyanocobaltate(III), potassium (ethylenediaminetetraacetato)cobaltate(III) dihydrate, hydridotetracarbonylcobalt(I), dicarbonyl(cyclopentadienyl)cobalt(I), octacarbonyldicobalt(O), hexacarbonyl(acetylene)dicobalt(O), bis(cyclopentadienyl)cobalt(I), (cyclopentadienyl)(1,5-cyclooctadiene)cobalt(I) and the like. Among these, particularly preferable compounds can be exemplified by cobalt chloride, cobalt nitrate, cobalt sulfate and the like.
Illustrative examples of the complex ion containing cobalt include pentaammineaquacobalt ion, nitropentaamminecobalt ion, dichlorotetraamminecobalt ion, dinitrotetraamminecobalt ion, carbonatotetraamminecobalt ion, tetranitrodiamminecobalt ion, hexanitrocobalt ion, tris(ethylenediamine)cobalt ion, dichlorobis(ethylenediamine)cobalt ion, tris(oxalato)cobalt ion, hexacyanocobalt ion, (ethylenediaminetetraacetato)cobalt ion and the like.
Of the said heavy metals, the compound containing silver, the complex ion containing silver or the silver ion preferably has a concentration in the medium of 10xe2x88x928M-10xe2x88x921M, and it is further preferable to adjust the concentration to be in the range of 10xe2x88x927M to 10xe2x88x922M. The compound containing cobalt, the complex ion containing cobalt or the cobalt ion preferably has a concentration in the medium of 10xe2x88x926-10xe2x88x921M, and it is further preferable to adjust the concentration to be in the range of 10xe2x88x925 to 10xe2x88x922M.
So far, there are no cases reported wherein the tissue culture of a plant producing a taxane-type diterpene is carried out in the presence of a compound containing silver, a complex ion containing silver or silver ion as an additive to the medium. Although compounds containing cobalt, or cobalt ions are contained as one of the medium components for such a medium that is generally used as a medium for the tissue culture of a plant belonging to genus Taxus, such as Linsmaier-Skoog medium, Murashige-Skoog medium, and Gamborg""s B-5 medium, they are used at a concentration of 1xc3x9710xe2x88x927M-4xc3x9710xe2x88x927M [Growth and breeding of a wo6dy plant, edited by the latest biotechnology complete works editors committee, Nogyo Tosho, P265-268], which is a much lower concentration than those used in the method of the present invention. In the meantime, there are no cases reported wherein the tissue culture of a plant producing a taxane-type diterpene is carried out in the presence of a compound containing cobalt or cobalt ions of such a high concentration that is used in the first invention of the present application, just like the case with the above-mentioned silver compound. In addition to that, it was beyond all expectations that the amount of the taxane-type diterpene to be produced is increased by the culture carried out in the presence of such heavy metals.
According to the first invention of the present application, by amines we refer to an amine or a salt thereof. As the amines, which are the objects for the first invention of the present application, both monoamines and polyamines can be used, however, the use of polyamines is particularly preferable.
In addition to that, examples of the amines, which are the objects for the first invention of the present application, include mono, di or trialkyl amines wherein a part of hydrogen atoms in the alkyl group may be substituted by hydroxyl group, such as methyl amine, ethyl amine, dimethyl amine, diethyl amine, triethyl amine, diethanol amine, triethanol amine or a salt thereof; polymethylene diamine wherein the polymethylene moiety may be interrupted by imino group, and H in the amino group can be substituted by lower alkyl group, such as putrescine, cadaverine, spermidine, spermin, ethylenediamine, N,N-diethyl-1,3-propane diamine, triethylene tetramine, or a salt thereof; cyclic alkyl amine such as cyclopentyl amine, cyclohexyl amine or a salt thereof, or a cyclic amine such as methenamine and piperazine, or a salt thereof. Among these amines, preferable amines can be exemplified by polyamines such as putrescine [NH2(CH2)4NH2], cadaverine [NH2(CH2)5NH2], spermidine [NH2(CH2)3NH(CH2)4NH2], spermin [NH2(CH2)3NH(CH2)4NH(CH2)3NH2], ethylene diamine [NH2(CH2)2NH2], N,N-diethyl-1,3-propane diamine [(C2H5)2N(CH2)3NH2], diethylene triamine [NH2(CH2)2NH(CH2)2NH2] and the like or a salt thereof.
The said amines preferably have a concentration in the medium of 10xe2x88x928M-10xe2x88x921M, and it is further preferable to adjust the concentration to be in the range of 10xe2x88x927M to 10xe2x88x922M.
One illustrative example wherein a secondary metabolite is shown to be induced by addition of amines to the plant tissue cultures is shown in Japanese Patent Laid-Open Publication No. 4-262788 wherein indole alkaloid production is shown to be induced by addition of amines to cultured cells of Catharanthus roseus. However, there are no cases reported wherein the tissue culture of a plant producing taxane-type diterpene, which is a different plant species from that of Catharanthus roseus, was carried out in the presence of amines as an additive to the medium, and it was beyond all expectations that the amount of the taxane-type diterpene, which has a totally different biosynthetic pathway from that of the indole alkaloid, to be produced can be increased thereby.
The antiethylene agent, which is an object for the first invention of the present application is not particularly limited to any specific substance as far as it is a substance which inhibits the ethylene biosynthesis mechanism of the cultures and/or a substance which removes the ethylene remaining in the cultures or existing in the gas phase or in the medium in the culture vessel containing the cultures.
Illustrative examples of a method to inhibit the ethylene biosynthesis mechanism include a method of inhibiting the activity of an enzyme which catalyzes the conversion of S-adenosylmethionine into 1-aminocyclopropane-1-carboxylic acid, and a method of inhibiting the activity of an enzyme which catalyzes the conversion of 1-aminocyclopropane-1-carboxylic acid into ethylene, and illustrative examples of the compound having the former function include, aminoxyacetic acid, acetylsalicylic acid, Rhizobitoxine, aminoethoxyvinylglycine, methoxyvinylglycine, a-aminoisobutyric acid, 2,4-dinitrophenol and the like. They can also include a salt, an ester, an amino acid derivative and a carbohydrate derivative of the said compound.
Illustrative examples of the salt include sodium, potassium, calcium, and magnesium salts, illustrative examples of the ester include methyl, ethyl, propyl, and butyl esters, illustrative examples of the amino acid derivatives include glycine, methionine, and phenylalanine derivatives, and illustrative examples of the carbohydrate derivative include glucose and maltose derivatives. The salt, ester, amino acid derivative, carbohydrate derivative according to the present invention are not limited to the above-mentioned compounds.
Illustrative examples of the compound having the latter function include gallic acid, a salt, an ester, an amino acid derivative and a carbohydrate derivative thereof [Hiroshi Hyodo, Society of Horticulture Autumn Convention 1987 Symposium Summary, p. 122, Susumu Kuraishi, Phytohormone, Tokyo University Publication, p.111].
Illustrative examples of the salt include sodium, potassium, calcium, and magnesium salts, illustrative examples of the ester include methyl, ethyl, propyl, and butyl esters, illustrative examples of the amino acid derivatives include glycine, methionine, and phenylalanine derivatives, and illustrative examples of the carbohydrate derivatives include glucose and maltose derivatives. The salt, ester, amino acid derivative, carbohydrate derivative according to the present invention are not limited to the above-mentioned compounds.
Illustrative examples of the substance which removes the ethylene remaining in the cultures or existing in the gas phase or the medium in the culture vessel containing the cultures include 1,5-cyclooctadiene and isothiocyanic acid, a salt, an ester (such as allyl isothiocyanate and benzyl isothiocyanate), an amino acid derivative and a carbohydrate derivative thereof [Megumi Munakata, Chemical control in plants, 29(1), 89-93 (1994)].
Illustrative examples of the salt include sodium, potassium, calcium, and magnesium salts, illustrative examples of the ester include methyl, ethyl, propyl, butyl, and allyl esters, illustrative examples of the amino acid derivatives include glycine, methionine, and phenylalanine derivatives, and illustrative examples of the carbohydrate derivatives include glucose and maltose derivatives. The salt, ester, amino acid derivative, carbohydrate derivative according to the present invention are not limited to the above-mentioned compounds.
The antiethylene agent is required to have a concentration in a culture medium of 10xe2x88x928M-10xe2x88x921M, and it is particularly preferable to control the concentration of the antiethylene agent to be in the range of 10xe2x88x927M to 10xe2x88x922M.
It is known that ethylene is one of phytohormones, and involved in various physiological phenomena caused in the plant, such as growth of individium, morphogenesis, and aging. A report by Kim, Dong II et al., Biotechnol. Bioeng., 38(4), 331-339 (1991) is an illustrative example wherein ethylene is utilized for improving the productivity of the secondary metabolite by the plant. However, in all the examples wherein controlling of ethylene is utilized for improving the productivity of the secondary metabolite, it is the control of ethylene supply to the plant tissue cultures, as typically shown in the above-mentioned report, and so far there have been no cases reported in which the control to inhibit the ethylene production is utilized to improve the production of the secondary metabolite, like the method of the present invention.
In addition to that, the antiethylene agent is generally utilized as a freshness keeping agent for flowers, fruits and vegetables, however, there have been no cases reported wherein the antiethylene agent is used for the purpose of improving the production of the secondary metabolite.
Under these circumstances, the present inventors ascertained that ethylene greatly inhibits the production of the taxane-type diterpene by the tissues and the cells of the taxane-type diterpene producing plant. Accordingly, based on the above-mentioned finding, the inventors cultured the said tissue cultures in the presence of the antiethylene agent, and found out that the antiethylene agent not only controls the above-mentioned inhibition but also remarkably improves the amount of the taxane-type diterpene resulting from the cultures. There have been no cases reported wherein the production of the taxane-type diterpene is induced by culturing the tissue cultures of a plant producing taxane-type diterpene in the presence of an antiethylene agent, and it was beyond all expectations that the productivity of the above-mentioned secondary metabolite can be even increased by the method of the first invention of the present application.
Examples of the medium to be used for the first invention of the present application include those known media which have been conventionally used for the plant tissue culture, such as medium of Murashige and Skoog (1962), medium of Linsmaier Skoog (1965), Woody Plant Medium (1981), Gamborg""s B-5 medium and Mitsui""s M-9 medium.
A phytohormone, and if necessary a carbon source, an inorganic component, vitamins, amino acids and the like may be added as well to these media.
As a carbon source, a disaccharide such as sucrose, maltose, and lactose, a monosaccharide such as glucose, fructose and galactose, starch or a mixture of two or more kinds of such sugar sources mixed at an appropriate ratio can be utilized.
As an inorganic component, illustrative examples include phosphorus, nitrogen, potassium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, copper, molybdenum, chlorine, sodium, iodine and cobalt, and these components can be added in the form of such a compound as potassium nitrate, sodium nitrate, calcium nitrate, potassium chloride, potassium monohydrogenphosphate, potassium dihydrogenphosphate, calcium chloride, magnesium sulfate, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, cobalt chloride and the like.
As the phytohormone, for example, auxin such as indoleacetic acid (IAA), naphthalenacetic acid (NAA), and 2,4-dichlorophenoxy acetic acid (2,4-D), and cytokinin such as kinetin, zeatin, and dihydrozeatin can be used.
As the vitamins, for example, biotin, thiamin (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, nicotinic acid and the like can be used.
As the amino acids, for example, glycine, phenylalanine, leucine, glutamine, cysteine and the like can be added.
Generally, the carbon source in a concentration of about 1-about 30 g/l, the inorganic component in a concentration of about 0.1 xcexcM-about 100 mM, the phytohormones in a concentration of about 0.01-about 10 xcexcM, and the vitamins and the amino acids respectively in a concentration of about 0.1-about 100 mg/l are used.
According to the present invention, both a liquid medium and such a solid medium that contains agar and gelan gum normally in an amount of 0.1-1% can be used, however, usually a liquid medium is preferable.
According to the tissue -culture of the present invention, a piece of a tissue or a cell of a root, a growing point, a leaf, a stem, a seed, a pollen, an anther and a calyx and the like of the said plant or cultured cells which are obtained by the tissue culture thereof in the above-mentioned medium or another conventional medium can be used.
The present invention can also be applied to neoplastic cell and/or hairy-root, obtained by infecting a plant tissue with Agrobacterium tumefaciens or Agrobacterium rhizogenes. 
By culturing these tissues or cells in the presence of at least one substance selected from the group consisting of jasmonic acids, compounds containing a heavy metal, complex ions containing a heavy metal, heavy metal ions, amines, and antiethylene agents, cultured tissues or cultured cells having higher taxane-type diterpene productivity than that of those obtained by the tissue culture carried out under the normal culture conditions, can be obtained.
When at least one compound selected from compounds containing a heavy metal, complex ions containing a heavy metal, heavy metal ions, amines, and antiethylene agents is used together with jasmonic acids represented by the above-mentioned general formulae (I), (II) or (III), the effect of the first inventiontof the present application can be enhanced.
Taxane-type diterpene can be fractionated from the cultures such as cultured tissues, cultured cells and culture medium, which are obtained according to the above-mentioned process, by extraction with an organic solvent such as methanol. It is also possible to recover the taxane-type diterpene continuously during culture by allowing an appropriate adsorbing agent or an organic solvent coexist in the culture medium.
One preferable example of the tissue culture according to the present invention can be illustrated as follows.
A piece of a plant body of a plant belonging to genus Taxus, such as a root, a growing point, a leaf, a stem, a seed and the like is sterilized and placed on Woody Plant Medium solidified with gelan gum, and kept at 10-35xc2x0 C. for about 14-60 days so that a part of the tissue piece is changed to callus. By subculturing the callus thus obtained, the growing speed is gradually increased and stabilized callus can be obtained. By the stabilized callus, we refer to a callus which remains in callus state during culture without showing differentiation into a shoot or a root and the cells of which have uniform growing speed.
Such stabilized callus is transferred to a liquid medium, suited for the growth, such as liquid Woody Plant Medium and grown. The growing speed is further increased in the liquid medium. According to the present invention, the stabilized callus or the cells constituting the above-mentioned callus are grown in a solid medium or a liquid medium in the presence of at least one substance selected from a group consisting of jasmonic acids, compounds containing a heavy metal, complex ions containing a heavy metal, heavy metal ions, amines and antiethylene agents. And, it is also possible to fractionate the stabilized callus or the cells constituting the said callus into a plurality of layers according to the difference in their specific gravities and grow the cells contained in at least one layer in a culture medium containing at least one substance selected from the group consisting of jasmonic acids, compounds containing a heavy metal, complex ions containing a heavy metal, heavy metal ions, amines and antiethylene agents.
In a generally known method to fractionate the cells according to their specific gravities, density gradient is formed by a medium for centrifugal separation, and the cells are layered over it, then centrifugal separation is carried out.
As a medium for centrifugal separation, Ficoll, Percoll (both produced by Pharmacia LKB Biotechnology Co. Ltd.,), sucrose and cesium chloride and the like are used. In the examples including Example No.5, the density gradient was produced by the use of Ficoll, however, the medium is not particularly limited to any substance as far as it does not damage the cells.
The number of the layers forming the density gradient is not particularly restricted. The difference between the specific gravities of layers is not particularly limited and each difference in the specific gravity can be the same or different.
Accordingly, the definition of the density gradient includes a case wherein the gradient changes continuously (the condition wherein the number of the layers forming the density gradient is close to infinite, and the specific gravity difference between each layer is close to 0).
The cells can be fractionated into a plurality of layers according to the difference in their specific gravities by thus forming the density gradient, layering the cells and carrying out the centrifugal separation.
The specific gravity of the layer to be formed is normally in the range of 1.00 to 1.20 g/ml, preferably in the range of 1.03 to 1.11 g/ml. As a layer to become an object for culture, at least one layer is selected, but it is also possible to select all the layers and culture them.
When a plurality-of layers are selected and cells contained in the selected layers are cultured, it is possible to culture the cells in these layers individually, but, it is also possible to mix the cells in two or more layers of the selected plurality of layers and culture them.
The cultured cells having high taxane-type diterpene productivity can be usually obtained by culturing cells contained in a layer having the specific gravity of 1.07 or less, but it is not always limited to this range, since it may fluctuate depending on the cells to be cultured or the culture conditions. There is also a tendency that the cells in a layer of a higher specific gravity, have a higher content of the taxane-type diterpene at the time when the fractionation is carried out according to the difference in the specific gravities. Accordingly, to ensure that cultured cells which produce the taxane-type diterpene at a high rate can be obtained, it is desirable that the cells in all the fractionated layers are cultured for a certain period, then the concentration of the taxane-type diterpene in the cells of each layer is measured, and the layer containing the cultured cells which produce the taxane-type diterpene at a high rate is selected from among those layers.
It is also possible to fractionate the cultured cells into a plurality of layers according to the difference in the specific gravities by preparing a medium for centrifugal separation having one particular specific gravity such as 1.07 g/ml, for example, and carrying out the centrifugal separation according to the above-mentioned method.
Furthermore, the first invention of the present application can be used together with the method of the second invention of the present application wherein the culture is carried out by controlling the oxygen concentration in a gas phase in a culture vessel to less than the oxygen concentration in the atmosphere, from the initial stage of the culture, or by controlling the dissolved oxygen concentration in a fluid medium which is in contact with the tissue or the cell to less than the saturated dissolved oxygen concentration at that temperature from the initial stage of the culture.
Here, by the initial stage of the culture, we refer to from the time when the culture was started through the 7th day after the start of the culture, and the controlling of the oxygen concentration in the gas phase in the culture vessel or the controlling of the dissolved oxygen concentration in the fluid medium which is in contact with the tissue or the cell is preferably done from the beginning of the culture. The controlling period is not particularly limited, and, the controlling under the said conditions can be done in the entire culture period, or only in a part of the entire culture period, however, it is preferable to carry out the control at least for 3 days during the entire culture period.
The oxygen concentration in the gas phase in the culture vessel is required to be controlled to 4-15%, and it is particularly preferable to control it to 6-12%. The dissolved oxygen concentration in the fluid medium is required to be controlled to 1-75% of the saturated dissolved oxygen concentration at that temperature and it is particularly preferable to control it to 10-75%.
It is also possible to combine the first invention of the present application, the second invention of the present application and the third invention of the present application all together.
According to the first invention of the present application, it is effective to add jasmonic acids when the cultured cells are in the exponential growth phase or in the stationary phase, and it is particularly preferable to add jasmonic acids in a transitional period from the exponential growth phase to the stationary phase. The same can be said of the timing of the treatment for increasing the amount of the endogenous jasmonic acids to be produced. For example, when cells are subcultured in every 21 days, the 7th-16th day is the suitable time for addition of the jasmonic acids or the treatment to increase the amount of the endogenous jasmonic acids to be produced, and when the cells in the exponential growth phase, for example those on the 7th-14th day are to be subcultured, the suitable time is immediately after the transplantation. The addition of the jasmonic acids or the treatment to increase the amount of the endogenous jasmonic acid to be produced can be done at a time, in a plurality of parts, or continuously.
It is effective to add compounds containing a heavy metal, complex ions containing a heavy metal or heavy metal ions after the beginning of the culture and before the transitional period of the cultured cells from the exponential growth phase to the stationary phase, and it is particularly preferable to add them at the beginning of the culture. The addition of the said compounds or the ions can be done at a time, or in a plurality of parts.
It is effective to add amines before the transitional period of the cells from the exponential growth phase to the stationary phase, and it is particularly preferable to add them at the beginning of the culture. The addition of said compounds can be done at a time or in a plurality of parts.
It is effective to add antiethylene agents before the transitional period of the cells from the exponential growth phase to the stationary phase, and it is particularly preferable to add them immediately after the transition to the stationary phase. The addition of the said compounds can be done at a time or in a plurality of parts.
The temperature for the tissue culture according to the first invention of the present application is usually about 10-about 35 xc2x0 C., and preferably about 23-28xc2x0 C. according to the high growing speed. As for the culture period, 14-42 days are preferable.
When a liquid medium is used for the culture according to the first invention of the present application, the cultured cells can be fractionated from the culture medium after the culture is completed, by such a method as decantation or filtration and the desired taxane-type diterpene can be fractionated from the cultured cells and/or the culture medium by such a method as extraction with an organic solvent
The second invention of the present application will be explained as follows.
According to the second invention of the present application, the culture of the plant means culture of a tissue or a cell of the plant, wherein the culture is carried out by a conventionally known process except that the culture is carried out by controlling the oxygen concentration in the gas phase of the culture vessel to below the atmospheric oxygen concentration from the initial stage of the culture, or by controlling the dissolved oxygen concentration in the fluid medium which is in contact with the tissue or the cell to below the saturated dissolved oxygen concentration at that temperature from the initial stage of the culture.
So far, in the culture of a plant producing the taxane-type diterpene, there has been no reports wherein the culture is carried out under such conditions that the oxygen concentration in the gas phase to be supplied to the culture vessel wherein the tissue or the cells are cultured or the dissolved oxygen concentration in the medium which is in contact with the tissue or the cells to below the atmospheric oxygen concentration or below the saturated dissolved oxygen concentration, and it was beyond all expectations that the amount of the taxane-type diterpene to be produced is increased by that.
According to the second invention of the present application, the oxygen concentration in the gas phase of the culture vessel wherein the tissue or the cells are cultured is required to be controlled to 4-15%, it is particularly preferably controlled to 6-12%. The dissolved oxygen concentration of the fluid medium which is in contact with the tissue or the cells is required to be controlled to 1-75% of the saturated dissolved oxygen concentration at that temperature, it is particularly preferably controlled to 10-75%.
Examples of a medium to be used in the second invention of the present application, include the medium conventionally known for the tissue culture of a plant, such as medium of Murashige and Skoog (1962), medium of Linsmaier Skoog (1965), Woody Plant Medium (1981), Gamborg""s B-5 medium, and Mitsui""s M-9 medium and the like.
A phytohormone, and if necessary a carbon source, an inorganic component, vitamins, amino acids and the like may be added as well to these media.
As a carbon source, a disaccharide such as sucrose, maltose, and lactose, a monosaccharide such as glucose, fructose and galactose, starch or a mixture of two or more kinds of such sugar sources mixed at an appropriate ratio can be utilized.
As an inorganic component, illustrative examples include phosphorus, nitrogen, potassium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, copper, molybdenum, chlorine, sodium, iodine and cobalt, and these components can be added in the form of such a compound as potassium nitrate, sodium nitrate, calcium nitrate, potassium chloride, potassium monohydrogenphosphate, potassium dihydrogenphosphate, calcium chloride, magnesium sulfate, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, cobalt chloride and the like.
As the phytohormone, for example, auxin such as indoleacetic acid (IAA), naphthalenacetic acid (NAA), and 2,4-dichlorophenoxy acetic acid (2,4-D), and cytokinin such as kinetin, zeatin, and dihydrozeatin can be used.
As the vitamins, for example, biotin, thiamin (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, nicotinic acid and the like can be used.
As the amino acids, for example, glycine, phenylalanine, leucine, glutamine, cysteine and the like can be added.
Generally, the carbon source in a concentration of about 1-about 30 g/l, the inorganic component in a concentration of about 0.1 xcexcM-about 100 mM, the phytohormones in a concentration of about 0.01-about 10 xcexcM, and the vitamins and the amino acids respectively in a concentration of about 0.1-about 100 mg/l are used.
According to the second invention of the present application, both a liquid medium and such a solid medium that contains agar and gelan gum normally in an amount of 0.1-1% can be used.
According to the tissue culture of the second invention of the present application, a piece of a tissue or a cell of a root, a growing point, a leaf, a stem, a seed, a pollen, an anther and a calyx and the like of the said plant or cultured cells which are obtained by the tissue culture thereof in the above-mentioned medium or another conventional medium can be used.
The second invention of the present application can also be applied to neoplastic cell and/or hairy-root, obtained by infection with Agrobacterium tumefaciens or Agrobacterium rhizogenes. 
When these tissues or cells are cultured by controlling the oxygen concentration in the gas phase in the culture vessel to less than the oxygen concentration in the atmosphere, from the initial stage of the culture, or by controlling the dissolved oxygen concentration in the fluid medium which is in contact with the tissue or the cell to less than the saturated dissolved oxygen concentration at that temperature, from the initial stage of the culture, cultured tissue or the cultured cells having higher taxane-type diterpene productivity than that of those obtained by the tissue culture carried out under normal culture conditions can be obtained.
According to the second invention of the present application, the initial stage of the culture refers to from the time when the culture was started through the 7th day after the start of the culture, and the controlling of the oxygen concentration in the gas phase in the culture vessel or the controlling of the dissolved oxygen concentration in the fluid medium which is in contact with the tissue or the cell is preferably done from the beginning of the culture.
The controlling period is not particularly limited, and, the controlling under the said condition can be done in the entire culture period, or only in a part of the entire culture period, however, it is preferable to carry out the control at least for 3 days during the entire culture period.
The production method according to the second invention of the present application can be used together with a culture method carried out in the presence of various kinds of taxane-type diterpene production promoting substances to further increase the productivity of the taxane-type diterpene.
Examples of the taxane-type diterpene production promoting substance include, for example, jasmonic acids represented by the above-mentioned general formulae (I), (II) or (III), compounds containing a heavy metal, complex ions containing a heavy metal, heavy metal ions, amines and antiethylene agents to be used for the above-mentioned first invention of the present application.
Also the second invention of the present application can be also used together with the method of the third invention of the present application which will be described later in detail, wherein the cells are fractionated into a plurality of layers according to the difference in their specific gravities, and the cells contained in at least one layer are cultured.
The production method according to the second invention of the present application can be used together with both the said method according to the first invention of the present application wherein culture is carried out in the presence of the jasmonic acids and the like and the method according to the third invention of the present application wherein the cells are fractionated into a plurality of layers according to the difference in their specific gravities and cells contained in at least one layer are cultured.
Taxane-type diterpene can be fractionated from the cultures such as cultured tissues, cultured cells and culture medium, which are obtained according to the above-mentioned process, by extraction with an organic solvent such as methanol.
One preferable example of the tissue culture according to the second invention of the present application can be illustrated as follows.
A piece of a plant body of a plant belonging to genus Taxus, such as a root, a growing point, a leaf, a stem, a seed and the like is sterilized and placed on Woody Plant Medium solidified with gelan gum, and kept at 10-35xc2x0 C. for about 14-60 days so that a part of the tissue piece is changed to callus. By subculturing the callus thus obtained, the growing speed is gradually increased and stabilized callus can be obtained. By the stabilized callus, we refer to a callus which remains in callus state during culture without showing differentiation into a shoot or a root and the cells of which have uniform growing speed.
Such stabilized callus is transferred to a liquid medium, suited for the growth, such as liquid Woody Plant Medium and grown. The growing speed is further increased in the liquid medium. According to the present invention, the stabilized callus or the cells constituting the above-mentioned callus is grown under the culture conditions wherein the oxygen concentration in a gas phase in a culture vessel is controlled to less than the oxygen concentration in the atmosphere from the initial stage of the culture, or the dissolved oxygen concentration in a fluid medium which is in contact with the tissue or the cell is controlled to less than the saturated dissolved oxygen concentration at that temperature, from the initial stage of the culture.
The tissue or the cell gains energy necessary for maintenance and growth of individium, by consuming oxygen (respiration). It is generally known that when a tissue or a cell is cultured, the cell mass is increased and the amount of the oxygen consumption is increased as well with the passage of culture period. Accordingly unless a ventilation gas is forcedly supplied from outside of the system, the oxygen concentration in the gas phase in the culture vessel such as a flask wherein the tissue or the cell is contained, or the dissolved oxygen concentration in the medium which is in contact with the tissue or the cell naturally decreases to a value less than the oxygen concentration in the atmosphere, or the saturated dissolved oxygen concentration at that temperature, with the passage of culture period.
The present invention is different from the above-mentioned finding on the point that the culture is carried out by actively controlling the oxygen concentration in the gas phase in the culture vessel which contains the tissue or the cell or the dissolved oxygen concentration in the culture medium to less than the oxygen concentration in the atmosphere or the saturated dissolved oxygen concentration at that temperature.
In one illustrative process to enhance the effect of the present invention, the oxygen concentration in the gas phase in the culture vessel or the dissolved oxygen concentration in the fluid medium is previously controlled to less than the oxygen concentration in the atmosphere or the saturated dissolved oxygen concentration at that temperature, prior to the subculture of the tissue or the cell in the culture vessel.
The controlling period is not particularly limited as mentioned above, however, it is preferable to carry out the control at least for 3 days during the entire culture period.
In addition to that, the controlling method is not particularly limited to any as far as it is a method wherein the oxygen concentration in the gas phase in the culture vessel which contains the tissue or the cell, or the dissolved oxygen concentration in the fluid medium which is in contact with the tissue or the cell, can be controlled to less than the oxygen concentration in the atmosphere or the saturated dissolved oxygen concentration at that temperature, and in some examples of such method, a gas having a controlled oxygen concentration, which is obtained by mixing an air with nitrogen and the like to lower the oxygen concentration, is directly sent into the gas phase in the culture vessel or the culture medium, or such a gas is directly sent into the culture medium outside of the culture vessel, i.e. in an aeration tank and the like, then the culture medium is poured into the culture vessel, or a gas such as air to be supplied to the culture vessel is directly sent into the gas phase or the culture medium by controlling the feed speed, or such a gas is directly sent into the culture medium outside of the culture vessel, i.e. in an aeration tank and the like then the culture medium is poured into the culture vessel, or the culture vessel is placed under low oxygen atmosphere to carry out culture or the culture is carried out in the presence of an oxygen adsorbing agent.
The temperature for the tissue culture according to the present invention is usually about 10-about 35xc2x0 C., and preferably about 23-28 xc2x0 C. according to the high growing speed. As for the culture period, 14-42 days are preferable.
When a liquid medium is used for the culture according to the present invention, the cultured cells can be fractionated from the culture medium after the culture is completed by such a method as decantation or filtration and the desired taxane-type diterpene can be fractionated from the cultured cells and/or the culture medium by such a method as extraction with an organic solvent. It is also possible to recover the desired compound continuously during the culture by allowing an adsorbing agent or an appropriate organic solvent coexist in the culture system.
The third invention of the present application will be explained as follows.
According to the third invention of the present application, a layer containing the cultured cells, which shall show high taxane-type diterpene productivity after being cultured, can be exemplified by a layer having the specific gravity of 1.07 or less.
In a generally known method to fractionate the cells according to their specific gravities, a density gradient is formed by a medium for centrifugal separation, and the cells are layered over it, then centrifugal separation is carried out.
As a medium for centrifugal separation, Ficoll, Percoll (both produced by Pharmacia LKB Biotechnology Co. Ltd.,), sucrose and cesium chloride and the like are used. In Examples, the density gradient was produced by the use of Ficoll, however, the medium is not particularly limited to any substance as far as it does not damage the cells. Ficoll has been used for separation of cell granules and the like (Hess, R. et al., Nature 208 (1965), 856-858) or separation of animal cells (Walder, I. A. et al., Proc. Soc. exptl. Biol. Med., 112(1963) 494-496) and the like.
The number of the layers forming the density gradient is not particularly limited.
In Examples, a density gradient wherein the difference of the specific gravity between each layer is 0.02 is formed by the layers having the specific gravity of 1.03, 1.05, 1.07, 1.09 and 1.11 (g/ml), however, the difference of the specific gravity is not limited to this value, and the difference of the specific gravity between each layer can be the same or different.
Accordingly, the definition of the density gradient includes a case wherein the gradient changes continuously (the condition wherein the number of the layers forming the density gradient is close to infinite, and the difference of the specific gravity between each layer is close to 0).
The cells can be fractionated into a plurality of layers according to the difference in their specific gravities by thus forming the density gradient, layering the cells and carrying out the centrifugal separation.
The specific gravity of the layer to be formed is normally in the range of 1.00 to 1.20 g/ml, preferably in the range of 1.03 to 1.11 g/ml. As a layer to become an object for culture, at least one layer is selected, but it is also possible to select all the layers and culture them.
When a plurality of layers are selected and cells contained in the selected layers are cultured, it is possible to culture the cells in these layers individually, but, it is also possible to mix the cells in two or more layers of the selected plurality of layers and culture them.
The cultured cells having high taxane-type diterpene productivity can be usually obtained by culturing cells contained in a layer having the specific gravity of 1.07 or less, but, it is not always limited to this range, since it may fluctuate depending on the cells to be cultured or the culture conditions. There is also a tendency that the cells in a layer of a higher specific gravity, have a higher content of the taxane-type diterpene at the time when the fractionation is carried out according to the difference in the specific gravities. Accordingly, to ensure that cultured cells which produce the taxane-type diterpene at a high rate can be obtained, it is desirable that the cells in all the fractionated layers are cultured for a certain period, then the concentration of the taxane-type diterpene in the cells of each layer is measured, and the layer containing the cultured cells which produce the taxane- type diterpene at a high rate is selected from among those layers.
So far, there have been no cases reported wherein the cultured cells of a plant producing the taxane-type diterpene are cultured after they are fractionated according to the specific gravity of the cells, and it was beyond all expectations that the cells can be fractionated into layers of cells each having different taxane-type diterpene productivity, by the difference of the specific gravities, and that the cells which produce the taxane-type diterpene at a high rate can be obtained by culture of cells which are contained in a layer having the specific gravity of 1.07 or less, and whose taxane-type diterpene content is not so high at the time when they are fractionated.
According to the present invention, it is also possible to fractionate the cultured cells into a plurality of layers according to the difference in the specific gravities by preparing a medium for centrifugal separation having one particular specific gravity such as 1.07 g/ml, for example, and carrying out the centrifugal separation according to the above-mentioned method.
The culture medium to be used for the present invention includes typical culture medium components. As such a component, an inorganic component and a carbon source are typically used, and phytohormones, vitamins, and if necessary, amino acids can be added as well. As a carbon source, a disaccharide such as sucrose, maltose, and lactose, monosaccharide such as glucose, fructose and galactose, starch or a mixture of two or more kinds of such sugar sources mixed at an appropriate ratio can be utilized.
As an inorganic component, illustrative examples include phosphorus, nitrogen, potassium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, copper, molybdenum, chlorine, sodium, iodine and cobalt, and these components can be added in the form of such a compound as potassium nitrate, sodium nitrate, calcium nitrate, potassium chloride, potassium monohydrogenphosphate, potassium dihydrogenphosphate, calcium chloride, magnesium sulfate, sodiumsulfate, ferrous sulfate, ferricsulfate, manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, cobalt chloride and the like.
As the phytohormone, for example, auxin such as indoleaceacid (IAA), naphthalenacetic acid (NAA), 2,4-dichlorophenoxy acetic acid (2,4-D), and cytokinin such as kinetin, zeatin, dihydrozeatin can be used.
As the vitamins, for example, biotin, thiamin (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, nicotinic acid and the like can be used.
As the amino acids, for example, glycine, phenylalanine, leucine, glutamine, cysteine and the like can be added.
Generally, the inorganic component in a concentration of about 0.1 xcexcM-about 100 mM, the carbon source in a concentration of about 1-about 30 g/l, the phytohormone in a concentration of about 0.01-about 10 xcexcM, and the vitamins and the amino acids respectively in a concentration of about 0.1-about 100 mg/l are used.
Examples of a medium to be used for the present invention include those known media which have been conventionally used for the plant tissue culture, such as Medium of Murashige and Skoog (1962), medium of Linsmaier Skoog (1965), Woody Plant Medium (1981), Gamborg""s B-5 medium and Mitsui""s M-9 medium to which the above-mentioned phytohormone, and if necessary, the above-mentioned carbon source, vitamins and amino acids are added.
According to the present invention, both a liquid medium and such a solid medium that contains agar and gelan gum normally in an amount of 0.1-1% can be used, however, usually a liquid medium is preferable.
According to the tissue culture of the present invention, a piece of a tissue or a cell of a root, a growing point, a leaf, a stem, a seed, a pollen, an anther and a calyx and the like of the said plant, or cultured cells which are obtained by the tissue culture thereof in the said medium or another conventional medium can be used.
By fractionating these cells into particular specific gravity ranges then culturing them according to the present invention, cultured cells having higher taxane-type diterpene productivity, in comparison with those in the control area wherein no fractionation was carried out, can be obtained. The taxane-type diterpene can be fractionated from these cultured cells by extraction with an organic solvent such as methanol.
One preferable example of the tissue culture according to the present invention can be illustrated as follows.
A piece of a plant body of a plant belonging to genus Taxus, such as a root, a growing point, a leaf, a stem, a seed and the like is sterilized and placed on Woody Plant Medium solidified with gelan gum, and kept at 10-35xc2x0 C. for 14-60 days so that a part of the tissue piece is changed to callus. By subculturing the callus thus obtained, the growing speed is gradually increased and stabilized callus can be obtained. By the stabilized callus, we refer to a callus which remains in callus state during culture without showing differentiation into a shoot or a root and the cells of which have uniform growing speed.
Such stabilized callus is transferred to a liquid medium, suited for the growth, such as liquid Woody Plant Medium and grown. The growing speed is further increased in the liquid medium.
The temperature for the tissue culture according to the present invention is usually about 10-about 35xc2x0 C., and preferably about 23-28xc2x0 C. according to the high growing speed. As for the culture period, 14-42 days are preferable.
When a liquid medium is used for the culture according to the present invention, the cultured cells can be fractionated from the culture medium after the culture is completed, by such a method as decantation or filtration and the desired taxane-type diterpene can be fractionated from this by such a method as extraction with an organic solvent.
According to the first invention and the second invention of the present application, taxane-type diterpene can be easily obtained in large quantity.
According to the third invention of the present application, cultured cells which produce taxane-type diterpene at a high rate can be obtained with a simple operation.
When the first, second or third invention of the present application is to be industrially executed, the efficiency can be further increased by employing the following fourth, fifth, sixth or seventh invention of the present application in an independent form or in a combined form.
That means, it is necessary to supply a gas containing oxygen to a culture liquid to culture tissues or cells of a plant which produces taxane-type diterpene. Normally, air is used for this purpose, however, after an intensive study, the present inventors found that the taxane-type diterpene production can be efficiently carried out by the use of a gas containing 0.03-10%, preferably 0.1-5% of carbon dioxide, as a gas to be introduced to a tank for culturing the tissues or the cells of the plant producing the taxane-type diterpene, and completed the fourth invention of the present application.
The present inventors also found that the productivity of the taxane-type diterpene in the cultures can be remarkably improved and the fluctuation of the taxane-type diterpene productivity due to the subculture can be controlled by carrying out a two-stage culture of the tissue or the cell of the plant producing the taxane-type diterpene, comprising a first stage using a medium to which an oxidizing agent or a water soluble organic compound containing oxygen is added for obtaining the tissues or the cells which is activated for production of the taxane-type diterpene in the subsequent stage, and a second stage which is carried out such conditions that promote the production of the taxane-type diterpene, and completed the fifth invention of the present application. Here, examples of the oxidizing agent include peroxodisulfates such as potassium peroxodisulfate and hydrogen peroxide, and examples of the water soluble organic compound containing oxygen include dimethyl formamide, dimethyl sulfoxide, and ethylene glycol and the like. The total concentration of the above-mentioned additive in the culture medium is preferably 10xe2x88x926M-10xe2x88x921M immediately after the addition, and it is further preferable to control the concentration to be in the range of 10xe2x88x925M to 10xe2x88x922M.
The present inventors also found that the high density culture of the tissue or the cell of the plant producing the taxane-type diterpene can be carried out by inoculating the tissues or the cells in a culture medium containing a saccharide in a concentration of 2-50 g/l, preferably 10-30 g/l, and/or nitrate ion in a concentration of 2-50 mmol/l, preferably 10-30 mmol/l, then by adding a nutrient source solution containing the saccharide in an amount of 0.2-5 g/l, preferably 0.5-3 g/l, and/or nitrate ion in an amount of 0.2-5 mmol/l, preferably 0.5-3 mmol/l per day with respect to the initial volume of the said culture medium, continuously or intermittently to the culture medium, thereby the taxane-type diterpene production volume per culture vessel can be remarkably increased and completed the sixth invention of the present application. Here, by the density, we refer to a cell mass per volume of the culture solution in the culture vessel, which is shown in terms of dry cell mass (g) per liter of the culture solution. According to the sixth invention of the present application, it is preferable to carry out culture while the culture medium is renewed by adding the nutrient source solution and simultaneously separating and removing the same volume of the medium from the tissues or the cells and to recover the taxane-type diterpene from at least one selected from the resulting cultures, the medium recovered by removal during the culture, and the medium obtained at the end of the culture. The sixth invention of the present application is particularly effective in improving the taxane-type diterpene productivity in the high density culture wherein the density of the tissue or the cell of the above-mentioned plant at the start of the culture with respect to the medium volume is 50 g fresh weight/l or higher.
Furthermore, though the culture is normally finished when the cells of high density are obtained, the present inventors achieved, through the intensive study, the continuous culture by continuing the culture while the cells are removed, and after further examination, finally completed a continuous culture method, which is the seventh invention of the present application. That means, the taxane-type diterpene can be produced with such a high rate that could be hardly attained with the conventional process, by adding the fresh medium continuously or intermittently in such a way that the specific renewing ratio defined by the dimensionless number F=VI/V/xcexc (wherein, V is the total volume of the culture medium in a culture tank, VI is the feed speed of the fresh medium, and xcexc is the specific growth rate of the tissues or the cells) is in the range of 0.1 to 10, and by recovering the taxane-type diterpene from the culture medium containing the tissues or the cells which is continuously or intermittently taken out from the tank and/or the culture solution which does not contain the tissue nor the cell and which is continuously or intermittently taken out from the tank, and completed the seventh invention of the present application. It is further preferable to set the specific renewing ratio of the culture medium, F, to 0.5-5. The saccharide concentration in the culture solution is preferably 5-40 g/l, and the nitrate ion concentration in the culture solution is preferably 10-40 mmol/l. The present invention can be effective with the cell density in terms of fresh cell weight per litter of 50-500 g, however, the higher the density is as far as it is in a range wherein extremely vigorous stirring is not required, the more efficiently the taxane-type diterpene can be produced, thus the preferable density is 200 g or higher per liter.
In order to combine the above-mentioned fourth, fifth, sixth, or seventh invention of the present application with the above-mentioned third invention of the present application, the cells obtained according to the third invention of the present application can be cultured according to the fourth, fifth, sixth or seventh invention of the present application to produce the desired taxane-type diterpene.